Douglas G. Storey

Pseudomonas aeruginosa Quorum-Sensing Systems May Control Virulence Factor Expression in the Lungs of Patients with Cystic Fibrosis

ABSTRACT Individuals with cystic fibrosis (CF) are commonly colonized with Pseudomonas aeruginosa. The chronic infections caused by P. aeruginosa are punctuated by acute exacerbations of the lung disease, which lead to significant morbidity and mortality. As regulators of virulence determinants, P. aeruginosa quorum-sensing systems may be active in the chronic lung infections associated with CF. We have examined the levels of autoinducer molecules and transcript accumulation from the bacterial populations found in the lungs of patients with CF. We detected biologically active levels of N-(3-oxododecanoyl)-l-homoserine (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL) in sputum from CF patients. Interestingly, it appears that C4-HSL is less frequently detected than 3-oxo-C12-HSL in the lungs of patients with CF. We also examined the transcription of the autoinducer synthase gene lasI and showed that it is frequently expressed in the lungs of patients with CF. We observed a significant correlation between the expression of lasI and four target genes of the Las quorum-sensing system. Taken together, our results indicate that quorum-sensing systems are active and may control virulence factor expression in the lungs of patients with CF.

Multidrug Efflux Pumps: Expression Patterns and Contribution to Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

ABSTRACT Pseudomonas aeruginosa biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance ofP. aeruginosa biofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexA andmexC in the developing biofilm. Remarkably, expression ofmexAB-oprM and mexCD-oprJ was not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprM andmexCD-oprJ expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosa biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosa biofilms.

Pseudomonas aeruginosa GacA, a factor in multihost virulence, is also essential for biofilm formation.

We have investigated a potential role for GacA, the response regulator of the GacA/GacS two‐component regulatory system, in Pseudomonas aeruginosa biofilm formation. When gacA was disrupted in strain PA14, a 10‐fold reduction in biofilm formation capacity resulted relative to wild‐type PA14. However, no significant difference was observed in the planktonic growth rate of PA14 gacA−. Providing gacA in trans on the multicopy vector pUCP‐gacA abrogated the biofilm formation defect. Scanning electron microscopy of biofilms formed by PA14 gacA− revealed diffuse clusters of cells that failed to aggregate into microcolonies, implying a deficit in biofilm development or surface translocation. Motility assays revealed no decrease in PA14 gacA− twitching or swimming abilities, indicating that the defect in biofilm formation is independent of flagellar‐mediated attachment and solid surface translocation by pili. Autoinducer and alginate bioassays were performed similarly, and no difference in production levels was observed, indicating that this is not merely an upstream effect on either quorum sensing or alginate production. Antibiotic susceptibility profiling demonstrated that PA14 gacA− biofilms have moderately decreased resistance to a range of antibiotics relative to PA14 wild type. This study establishes GacA as a new and independent regulatory element in P. aeruginosa biofilm formation.

Discerning the Complexity of Community Interactions Using a Drosophila Model of Polymicrobial Infections

A number of human infections are characterized by the presence of more than one bacterial species and are defined as polymicrobial diseases. Methods for the analysis of the complex biological interactions in mixed infections with a large number of microorganisms are limited and do not effectively determine the contribution of each bacterial species to the pathogenesis of the polymicrobial community. We have developed a novel Drosophila melanogaster infection model to study microbe–microbe interactions and polymicrobe–host interactions. Using this infection model, we examined the interaction of 40 oropharyngeal isolates with Pseudomonas aeruginosa. We observe three classes of microorganisms, one of which acts synergistically with the principal pathogen, while being avirulent or even beneficial on its own. This synergy involves microbe–microbe interactions that result in the modulation of P. aeruginosa virulence factor gene expression within infected Drosophila. The host innate immune response to these natural-route polymicrobial infections is complex and characterized by additive, suppressive, and synergistic transcriptional activation of antimicrobial peptide genes. The polymicrobial infection model was used to differentiate the bacterial flora in cystic fibrosis (CF) sputum, revealing that a large proportion of the organisms in CF airways has the ability to influence the outcome of an infection when in combination with the principal CF pathogen P. aeruginosa.

[25] The MBEC assay system: Multiple equivalent biofilms for antibiotic and biocide susceptibility testing

Publisher Summary A number of technologies have been developed to study biofilm growth. Although these technologies produce reproducible biofilms for the study of biofilm growth, structure, and physiology, they have not been amenable for the routine study of biofilm susceptibility to antibiotics and biocides. For this reason, virtually every antibiotic and biocide available has been selected for activity against planktonic organisms. These drugs often have been found to lack activity against microbial biofilms. The MBEC (minimum biofilm eradication concentration) Assay System using the Calgary Biofilm Device provides, for the first time, an assay easily applicable to screening antibiotics and biocides for activity against microbial biofilms. The MBEC Assay System is ideally suited either for screening new putative antibiotics and/or biocides, or for the determination of both the MIC (minimal inhibitory concentration) and MBEC values in clinical situations for the treatment of chronic, recurrent, or device-related infections. The MBEC Assay System produces 96 equivalent biofilms formed under flow conditions, without the need for pumps. Further, as it is based on the standard 96 well platform, it conforms to existing technology available in most laboratories. The MBEC Assay System consists of a two-piece disposable plastic apparatus used for biofilm formation.

Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster.

ABSTRACT The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.

Quorum-sensing mutations affect attachment and stability of Burkholderia cenocepacia biofilms.

ABSTRACT Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.

Rapid Colorimetric Assay for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa

ABSTRACT A colorimetric assay based on the reduction of a tetrazolium salt {2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT)} for rapidly determining the susceptibility of Pseudomonas aeruginosa isolates to bactericidal antibiotics is described. There was excellent agreement between the tobramycin and ofloxacin MICs determined after 5 h using the XTT assay and after 18 h using conventional methods. The data suggests that an XTT-based assay could provide a useful method for rapidly determining the susceptibility of P. aeruginosa to bactericidal antibiotics.

Regulation of toxA and regA by the Escherichia coli fur gene and identification of a Fur homologue in Pseudomonas aeruginosa PA103 and PA01

A multicopy plasmid containing the Escherichia coli fur gene was introduced into Pseudomonas aeruginosa strain PA103C. This strain contains a toxA‐lacZ fusion integrated into its chromosome at the toxA locus. Beta‐galactosidase synthesis in this strain is regulated by iron, as is seen for exotoxin A production. Beta‐galactosidase synthesis and exotoxin A production in PA103C containing multiple copies of E. coli fur was still repressed in low iron conditions. The transcription of regA, a positive regulator of toxA, was also found to be inhibited by multiple copies of the E. coli fur gene. In addition, the ability of PA103C containing multiple copies of E. coli fur to produce protease was greatly reduced relative to PA103C containing a vector control.

Mechanism of action of puroindoline derived tryptophan-rich antimicrobial peptides.

A tryptophan (Trp)-rich region in the wheat endosperm protein, puroindoline A, was previously shown to possess potent antimicrobial activity against Gram-positive and Gram-negative bacteria and this was attributed to the peptide inducing membrane instability. In the present work, the antimicrobial activity of the corresponding Trp-rich region in the puroindoline B isoform was examined and its antimicrobial activity was characterized. Unexpectedly, the puroindoline B Trp-rich peptide (PuroB) was relatively inactive compared to the related puroindoline A peptide (PuroA), despite strong sequence similarity. Using the sequence of PuroA as a template, a series of PuroB variants were synthesized and the antimicrobial activity was restored. Interestingly, all of these PuroB peptides preferentially interacted with negatively charged phospholipids, but unlike PuroA, they did not disrupt the integrity of lipid bilayers. This suggests that the primary mode of action of the PuroB peptides involves an antimicrobial target other than the bacterial membrane. Further tests revealed that all of the puroindoline derived peptides bind deoxyribonucleic acid (DNA) and block macromolecular synthesis in vivo. Based on these results, it appears that the interaction between puroindoline derived peptides and membranes is only an initial step in the mode of action and that binding to intracellular targets, such as DNA and ribonucleic acid (RNA), contributes significantly to their antimicrobial mode of action.